Purification of vaccinia virus-induced thymidine kinase activity from [ 35S]methionine-labeled cells

Abstract

Vaccinia virus thymidine kinase (TK) activity was purified from the cytosol of [ 35S]methionine-labeled, virus-infected LM(TK-) mouse fibroblast cells. The purification procedure entailed (i) selective binding of TK activity to a Sepharose 4B-5′-amino-5′-deoxythymidine gel and elution of the enzyme from the enzyme-gel complex, (ii) preparative polyacrylamide gel electrophoresis (PAGE), and (iii) glycerol gradient centrifugation of the purified vaccinia virus TK activity. [ 35S]Methionine-labeled cytosol fractions from uninfected cells were also mixed with nonlabeled vaccinia virus TK and purified by the same procedure to determine whether cellular proteins copurified with vaccinia virus TK. The purified vaccinia virus TK activity was adsorbed to an immunoadsorbent, made by coupling immunoglobulins (IgG) from antivaccinia virus TK rabbit antisera with CNBr-activated Sepharose 413. After elution of the labeled polypeptides from the immunoadsorbent, the eluates, as well as samples from earlier purification steps, were analyzed by SDS-polyacrylamide slab gel electrophoresis and autoradiography. Radioactive vaccinia virus TK that had been partially purified by analytical disc PAGE and by isoelectric focusing in polyacrylamide gels was also analyzed by SDS-polyacrylamide slab gel electrophoreses and autoradiography. The results of these experiments suggest that vaccinia virus TK consists of two subunits with molecular weights of about 40,000–42,000. Induction of the 40,000- to 42,000-dalton polypeptide was also observed in cells infected by vaccinia virus mutant 1004B, suggesting that this mutant induces the formation of an enzymatically inactive TK polypeptide.